Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag)hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on "active pairs" of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the fourpentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.The protein-DNA interactions that take place at eukaryotic origins of DNA replication are poorly characterized. This situation reflects, in part, the failure to identify DNA sequences that constitute higher eukaryotic origins of replication (13). Moreover, there is limited structural information about the initiator proteins that recognize origins of replication (35, 67). Indeed, structural information is currently limited to the origin binding domains of initiators encoded by simian virus 40 (SV40) (45), bovine papillomavirus (33), and Epstein-Barr virus (1, 2).In view of these limitations, a useful model for studies of the protein-DNA interactions that take place at eukaryotic origins is the binding of the virus-encoded T-antigen (T-ag) to the SV40 origin of replication. The well-characterized SV40 core origin is 64 bp long and consists of three separate domains (20,21). The central region, termed site II (or pentanucleotide palindrome), contains four GAGGC pentanucleotides that serve as binding sites for T-ag (24, 69, 70). Site II is flanked by a 17-bp adenine-thymine (AT)-rich domain and the early palindrome (EP) (reviewed in references 4 and 29).T-ag, a 708-amino-acid phosphoprotein, has been extensively studied (reviewed in references 4, 9, and 29). The structure of the T-ag domain that is necessary and sufficient for binding to the SV40 origin, T-ag-obd , was solved by use of nuclear magnetic resonance techniques (45). When the structure was viewed in terms of previous mutagenesis studies of T-ag (65, 76), considerable insight into the mechanism of binding of T-ag to individual pentanucleotides was obtained. For example, it is now apparent that site-specific binding is mediated by a pair of loops (45), a common motif in protein-DNA interactions (9, 41). Additional insights into T-ag binding to individual pentanucleotides, ...

To better define protein-DNA interactions at a eukaryotic origin, the domain of simian virus 40 (SV40) large T antigen that specifically interacts with the SV40 origin has been purified and its binding to DNA has been characterized. Evidence is presented that the affinity of the purified T antigen DNA-binding domain for the SV40 origin is comparable to that of the full-length T antigen. Furthermore, stable binding of the T antigen DNA-binding domain to the SV40 origin requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix and positioned in a head-to-head orientation. Although two pairs of pentanucleotides are present in the SV40 origin, footprinting and band shift experiments indicate that binding is limited to dimer formation on a single pair of pentanucleotides. Finally, it is demonstrated that the T antigen DNA-binding domain interacts poorly with single-stranded DNA.

The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd131–260) have been determined. Binding of the purified T-ag-obd131–260 is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.